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1.  Epstein-Barr Virus BART9 miRNA Modulates LMP1 Levels and Affects Growth Rate of Nasal NK T Cell Lymphomas 
PLoS ONE  2011;6(11):e27271.
Nasal NK/T cell lymphomas (NKTCL) are a subset of aggressive Epstein-Barr virus (EBV)-associated non-Hodgkin's lymphomas. The role of EBV in pathogenesis of NKTCL is not clear. Intriguingly, EBV encodes more than 40 microRNAs (miRNA) that are differentially expressed and largely conserved in lymphocryptoviruses. While miRNAs play a critical role in the pathogenesis of cancer, especially lymphomas, the expression and function of EBV transcribed miRNAs in NKTCL are not known. To examine the role of EBV miRNAs in NKTCL, we used microarray profiling and qRT-PCR to identify and validate expression of viral miRNAs in SNK6 and SNT16 cells, which are two independently derived NKTCL cell lines that maintain the type II EBV latency program. All EBV BART miRNAs except BHRF-derived miRNAs were expressed and some of these miRNAs are expressed at higher levels than in nasopharyngeal carcinomas. Modulating the expression of BART9 with antisense RNAs consistently reduced SNK6 and SNT16 proliferation, while antisense RNAs to BARTs-7 and -17-5p affected proliferation only in SNK6 cells. Furthermore, the EBV LMP-1 oncoprotein and transcript levels were repressed when an inhibitor of BART9 miRNA was transfected into SNK6 cells, and overexpression of BART9 miRNA increased LMP-1 protein and mRNA expression. Our data indicate that BART9 is involved in NKTCL proliferation, and one of its mechanisms of action appears to be regulating LMP-1 levels. Our findings may have direct application for improving NKTCL diagnosis and for developing possible novel treatment approaches for this tumor, for which current chemotherapeutic drugs have limited effectiveness.
PMCID: PMC3213120  PMID: 22102884
2.  Epstein–Barr virus microRNAs and lung cancer 
British Journal of Cancer  2011;105(2):320-326.
We conducted the first analysis of viral microRNAs (miRNAs) in lung cancer, with a focus on Epstein–Barr virus (EBV).
We evaluated viral miRs with a two-channel oligo-array targeting mature, anti-sense miRNAs in 290 cases. In 48 cases, we compared microarray and real-time quantitative PCR (qPCR) expression for three EBV miRNAs. We tested for EBV DNA, RNA, and protein in tumour tissue from six cases with and six cases without strong qPCR-based evidence of EBV miRNAs.
The EBV miRNAs strongly differentiated between adenocarcinoma and squamous cell carcinoma using the microarray (P<0.01 for 9 out of 16 EBV miRNAs). However, microarray and qPCR measurements of BART1, BART2, and BHRF1–3 expression were not significantly correlated (P=0.53, 0.94, and 0.47, respectively). Although qPCR provided substantial evidence of EBV miRNAs in 7 out of 48 cases, only 1 of these 7 cases had detectable EBV DNA in tumour tissue. None had detectable EBV RNA or protein by histochemical stains.
In a comprehensive evaluation of EBV miRNA, DNA, RNA, and protein in lung cancer, we found little evidence of EBV in lung tumour tissue. Discrepancies between microarray- and qPCR-based strategies highlight the difficulty of validating molecular markers of disease. Our results do not support a role of EBV in lung cancer.
PMCID: PMC3142804  PMID: 21654679
Epstein–Barr virus; lung cancer; microRNA; microarray; qPCR
3.  Expression of Adipose MicroRNAs Is Sensitive to Dietary Conjugated Linoleic Acid Treatment in Mice 
PLoS ONE  2010;5(9):e13005.
Investigation of microRNAs (miRNAs) in obesity, their genetic targets and influence by dietary modulators is of great interest because it may potentially identify novel pathways involved in this complex metabolic disorder and influence future therapeutic approaches. This study aimed to determine whether miRNAs expression may be influenced by conjugated linoleic acid (CLA), currently used to induce fat loss.
Methodology/Principal Findings
We determined retroperitoneal adipose tissue (rWAT) expression of five miRNAs related to adipocyte differentiation (miRNA-143) and lipid metabolism (miRNA-103 and -107) and altered in obesity (miRNA-221 and -222), using the TaqMan®MicroRNA Assay (Applied-Biosystems). In the first experiment, mice were fed with a standard fat diet and orally treated with sunflower oil (control group) and 3 or 10 mg CLA/day for 37 days. In the second experiment, mice were fed with a high fat diet for 65 days. For the first 30 days, mice received the same doses of CLA described above and, from that time onwards, animals received a double dose. Results showed that expression of selected miRNAs was modified in response to CLA treatment and metabolic status. Interestingly, a strong correlation was observed between miR-103 and -107 expression, as well as miR-221 and -222 in both experiments. Moreover, changes in miRNAs expression correlated with several adipocyte gene expressions: miR-103 and -107 correlated with genes involved in fatty acid metabolism whereas miR-221 and miR-222 correlated with the expression of adipocytokines. Regarding the minor changes observed in miR-143 expression, no differences in expression of adipogenic markers were observed.
Although elucidating the functional implications of miRNAs is beyond the scope of this study, these findings provide the first evidence that miRNAs expression may be influenced by dietary manipulation, reflecting or even contributing to the new metabolic state originated by CLA treatment.
PMCID: PMC2946340  PMID: 20886002
4.  MicroRNA Alterations of Pancreatic Intraepithelial Neoplasms (PanINs) 
Clinical Cancer Research  2011;18(4):981-992.
MicroRNA alterations are likely to contribute to the development of pancreatic cancer and may serve as markers for the early detection of pancreatic neoplasia.
Experimental Design
To identify the microRNA alterations that arise during the development of pancreatic cancer we determined the levels of 735 miRNAs in 34 pancreatic intraepithelial neoplasias (PanINs) and 15 normal pancreatic duct samples isolated by laser capture microdissection using TaqMan miRNA microarrays. Differential expression of selected miRNAs was confirmed by fluorescent in-situ hybridization analysis and by qRT-PCR analysis of selected candidate microRNAs in an independent set of PanIN and normal duct samples.
We identified 107 aberrantly expressed miRNAs in different PanIN grades compared with normal pancreatic duct samples, and 35 aberrantly expressed miRNAs in PanIN-3 lesions compared with normal pancreatic duct samples. These differentially expressed miRNAs included those that have been previously identified as differentially expressed in pancreatic ductal adenocarcinomas (including miR-21, miR-200a/b/c, miR-216a/b, miR-217, miR-146a, miR-155, miR-182, miR-196b, miR-203, miR-222, miR-338-3p, miR-486-3p and others) as well as miRNAs not previously described as differentially expressed in these lesions (miR-125b, miR-296-5p, miR-183*, miR-603, miR-625/*, miR-708 and others). MiR-196b was the most selectively differentially expressed miRNA in Panin-3 lesions.
Many miRNAs undergo aberrant expression in PanIN lesions and are likely to be important in the development of pancreatic ductal adenocarcinoma. MicroRNAs such as miR-196b whose expression is limited to PanIN-3 lesions or pancreatic cancers could be useful as diagnostic markers.
PMCID: PMC3288338  PMID: 22114139
5.  Sex differences in the expression of lupus-associated miRNAs in splenocytes from lupus-prone NZB/WF1 mice 
A majority of autoimmune diseases, including systemic lupus erythematosus (SLE), occur predominantly in females. Recent studies have identified specific dysregulated microRNAs (miRNAs) in both human and murine lupus, implying an important contribution of these miRNAs to lupus pathogenesis. However, to date, there is no study that examined sex differences in miRNA expression in immune cells as a plausible basis for sex differences in autoimmune disease. This study addresses this aspect in NZB/WF1 mice, a classical murine lupus model with marked female bias, and further investigates estrogen regulation of lupus-associated miRNAs.
The Taqman miRNA assay system was used to quantify the miRNA expression in splenocytes from male and female NZB/WF1 mice at 17–18, 23, and 30 weeks (wks) of age. To evaluate potential estrogen's effect on lupus-associated miRNAs, 6-wk-old NZB/WF1 male mice were orchidectomized and surgically implanted with empty (placebo) or estrogen implants for 4 and 26 wks, respectively. To assess the lupus status in the NZB/WF1 mice, serum anti-dsDNA autoantibody levels, proteinuria, and renal histological changes were determined.
The sex differences in the expression of lupus-associated miRNAs, including the miR-182-96-183 cluster, miR-155, miR-31, miR-148a, miR-127, and miR-379, were markedly evident after the onset of lupus, especially at 30 wks of age when female NZB/WF1 mice manifested moderate to severe lupus when compared to their male counterparts. Our limited data also suggested that estrogen treatment increased the expression of aforementioned lupus-associated miRNAs, with the exception of miR-155, in orchidectomized male NZB/WF1 mice to a similar level in age-matched intact female NZB/WF1 mice. It is noteworthy that orchiectomy, itself, did not affect the expression of lupus-associated miRNAs.
To our knowledge, this is the first study that demonstrated sex differences in the expression of lupus-associated miRNAs in splenocytes, especially in the context of autoimmunity. The increased expression of lupus-associated miRNA in female NZB/WF1 mice and conceivably in estrogen-treated orchidectomized male NZB/WF1 mice was associated with lupus manifestation. The notable increase of lupus-associated miRNAs in diseased, female NZB/WF1 mice may be a result of both lupus manifestation and the female gender.
PMCID: PMC3843556  PMID: 24175965
Sex differences; Lupus; microRNA; Estrogen; Splenocytes; NZB/WF1
6.  Circulating miR-150 and miR-342 in plasma are novel potential biomarkers for acute myeloid leukemia 
MicroRNAs (miRNAs) are small (19-22-nt) single-stranded noncoding RNA molecules whose deregulation of expression can contribute to human disease including the multistep processes of carcinogenesis in human. Circulating miRNAs are emerging biomarkers in many diseases and cancers such as type 2 diabetes, pulmonary disease, colorectal cancer, and gastric cancer among others; however, defining a plasma miRNA signature in acute myeloblastic leukemia (AML) that could serve as a biomarker for diagnosis or in the follow-up has not been done yet.
TaqMan miRNA microarray was performed to identify deregulated miRNAs in the plasma of AML patients. Quantitative real-time RT-PCR was used to validate the results. Receiver-operator characteristic (ROC) curve analysis was conducted to evaluate the diagnostic accuracy of the highly and significantly identified deregulated miRNA(s) as potential candidate biomarker(s).
The plasma expression level of let-7d, miR-150, miR-339, and miR-342 was down-regulated whilst that of let-7b, and miR-523 was up-regulated in the AML group at diagnosis compared to healthy controls. ROC curve analyses revealed an AUC (the areas under the ROC curve) of 0.835 (95% CI: 0.7119– 0.9581; P<0.0001) and 0.8125 (95% CI: 0.6796–0.9454; P=0.0005) for miR-150, and miR-342 respectively. Combined ROC analyses using these 2 miRNAs revealed an elevated AUC of 0.86 (95% CI: 0.7819–0.94; P<0.0001) indicating the additive effect in the diagnostic value of these 2 miRNAs. QRT-PCR results showed that the expression level of these two miRs in complete remission AML patients resembled that of healthy controls.
Our findings indicated that plasma miR-150 and miR-342 are novel important promising biomarkers in the diagnosis of AML. These novel and promising markers warrant validation in larger prospective studies.
PMCID: PMC3579719  PMID: 23391324
AML; Micro-RNA; Plasma; Biomarker
7.  Real-time quantification of microRNAs by stem–loop RT–PCR 
Nucleic Acids Research  2005;33(20):e179.
A novel microRNA (miRNA) quantification method has been developed using stem–loop RT followed by TaqMan PCR analysis. Stem–loop RT primers are better than conventional ones in terms of RT efficiency and specificity. TaqMan miRNA assays are specific for mature miRNAs and discriminate among related miRNAs that differ by as little as one nucleotide. Furthermore, they are not affected by genomic DNA contamination. Precise quantification is achieved routinely with as little as 25 pg of total RNA for most miRNAs. In fact, the high sensitivity, specificity and precision of this method allows for direct analysis of a single cell without nucleic acid purification. Like standard TaqMan gene expression assays, TaqMan miRNA assays exhibit a dynamic range of seven orders of magnitude. Quantification of five miRNAs in seven mouse tissues showed variation from less than 10 to more than 30 000 copies per cell. This method enables fast, accurate and sensitive miRNA expression profiling and can identify and monitor potential biomarkers specific to tissues or diseases. Stem–loop RT–PCR can be used for the quantification of other small RNA molecules such as short interfering RNAs (siRNAs). Furthermore, the concept of stem–loop RT primer design could be applied in small RNA cloning and multiplex assays for better specificity and efficiency.
PMCID: PMC1292995  PMID: 16314309
8.  MicroRNA-134 as a potential plasma biomarker for the diagnosis of acute pulmonary embolism 
Acute pulmonary embolism (APE) remains a diagnostic challenge due to a variable clinical presentation and the lack of a reliable screening tool. MicroRNAs (miRNAs) regulate gene expression in a wide range of pathophysiologic processes. Circulating miRNAs are emerging biomarkers in heart failure, type 2 diabetes and other disease states; however, using plasma miRNAs as biomarkers for the diagnosis of APE is still unknown.
Thirty-two APE patients, 32 healthy controls, and 22 non-APE patients (reported dyspnea, chest pain, or cough) were enrolled in this study. The TaqMan miRNA microarray was used to identify dysregulated miRNAs in the plasma of APE patients. The TaqMan-based miRNA quantitative real-time reverse transcription polymerase chain reactions were used to validate the dysregulated miRNAs. The receiver-operator characteristic (ROC) curve analysis was conducted to evaluate the diagnostic accuracy of the miRNA identified as the candidate biomarker.
Plasma miRNA-134 (miR-134) level was significantly higher in the APE patients than in the healthy controls or non-APE patients. The ROC curve showed that plasma miR-134 was a specific diagnostic predictor of APE with an area under the curve of 0.833 (95% confidence interval, 0.737 to 0.929; P < 0.001).
Our findings indicated that plasma miR-134 could be an important biomarker for the diagnosis of APE. Because of this finding, large-scale investigations are urgently needed to pave the way from basic research to clinical utilization.
PMCID: PMC3189884  PMID: 21943159
9.  The microRNAs of Epstein-Barr Virus are expressed at dramatically differing levels among cell lines 
Virology  2009;386(2):387-397.
Epstein-Barr Virus (EBV) encodes multiple microRNAs (miRNAs) from two primary transcripts, BHRF1 and the BARTs. The expression of BHRF1 miRNAs is dependent on the type of viral latency, whereas the BART miRNAs are expressed in cells during all forms of latency. It is not known how these miRNAs are otherwise regulated, though. We have used quantitative, stem-loop, real-time PCR to measure the expression of EBV’s miRNAs and found them to differ nearly 50- and 25-fold among all tested cell lines and among EBV-positive Burkitt’s lymphomas, respectively. In addition, the expression of individual BART miRNAs within a cell can differ by 50-fold or more despite the fact these miRNAs are likely transcribed together as a single primary transcript. These measurements are illuminating: they indicate that few of EBV’s miRNAs are expressed at levels comparable to those of cellular miRNAs in most cell lines and therefore likely function interdependently.
PMCID: PMC2763627  PMID: 19217135
Epstein-Barr Virus; microRNAs; BamHI A rightward transcripts; stem-loop real-time PCR
10.  MicroRNAs and in kidney function and disease 
MicroRNAs (miRNA) are short non-coding RNA sequences that regulate gene expression by blocking protein translation or inducing mRNA degradation. miRNA is found in various tissues with variable expression and changes in expression are related to various disease processes. Evidence suggests that changes in miRNA expression are critical for the normal development of kidney tissue. Alternatively, in diseases such as diabetic nephropathy, polycystic kidney disease, and lupus nephritis, specific miRNAs may enhance disease manifestations in a myriad of ways, ranging from activation of fibrotic pathways to anatomical changes that abet proteinuria. The variable expression of miRNA in kidney tissue, whether in the context of normal development or disease processes, makes miRNAs a valuable new tool for understanding, diagnosing, and discovering therapeutic options for pathological processes that affect the kidney.
PMCID: PMC3062898  PMID: 21420034
11.  MicroRNAs Implicated in the Immunopathogenesis of Lupus Nephritis 
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the deposition of immune complexes due to widespread loss of immune tolerance to nuclear self-antigens. Deposition in the renal glomeruli results in the development of lupus nephritis (LN), the leading cause of morbidity and mortality in SLE. In addition to the well-recognized genetic susceptibility to SLE, disease pathogenesis is influenced by epigenetic regulators such as microRNAs (miRNAs). miRNAs are small, noncoding RNAs that bind to the 3′ untranslated region of target mRNAs resulting in posttranscriptional gene modulation. miRNAs play an important and dynamic role in the activation of innate immune cells and are critical in regulating the adaptive immune response. Immune stimulation and the resulting cytokine milieu alter miRNA expression while miRNAs themselves modify cellular responses to stimulation. Here we examine dysregulated miRNAs implicated in LN pathogenesis from human SLE patients and murine lupus models. The effects of LN-associated miRNAs in the kidney, peripheral blood mononuclear cells, macrophages, mesangial cells, dendritic cells, and splenocytes are discussed. As the role of miRNAs in immunopathogenesis becomes delineated, it is likely that specific miRNAs may serve as targets for therapeutic intervention in the treatment of LN and other pathologies.
PMCID: PMC3741610  PMID: 23983769
12.  The role of promoter methylation in Epstein-Barr virus (EBV) microRNA expression in EBV-infected B cell lines 
Experimental & Molecular Medicine  2011;43(7):401-410.
Epstein-Barr virus (EBV) microRNAs (miRNAs) are expressed in EBV-associated tumors and cell lines, but the regulation mechanism of their expression is unclear yet. We investigated whether the expression of EBV miRNAs is epigenetically regulated in EBV-infected B cell lines. The expression of BART miRNAs was inversely related with the methylation level of the BART promoter at both steady-state and following 5-aza-2'-deoxycytidine treatment of the cells. The expression of BHRF1 miRNAs also became detectable with the demethylation of Cp/Wp in latency I EBV-infected cell lines. Furthermore, in vitro methylation of the BART and Cp promoters reduced the promoter-driven transactivation. In contrast, tricostatin A had little effect on the expression of EBV miRNA expression as well as on the BART and Cp/Wp promoters. Our results suggest that promoter methylation, but not histone acetylation, plays a role in regulation of the EBV miRNA expression in EBV-infected B cell lines.
PMCID: PMC3158499  PMID: 21628990
decitabine; DNA methylation; Herpesvirus 4, human; microRNAs; promoter regions, genetic
13.  Differentially expressed microRNAs in the serum of cervical squamous cell carcinoma patients before and after surgery 
The purpose of this study was to detect the serum microRNAs (miRNAs) that are differentially expressed in cervical squamous cell carcinoma (SCC) patients and negative controls, with a focus on the miRNA profiles of the patients before and after surgery. The aim of the study is to evaluate the potential of these miRNAs as novel markers for the post-therapeutic monitoring of cervical SCC patients.
A total of 765 serum miRNAs from 10 cervical SCC patients before surgery, 10 cervical SCC patients after surgery, and 10 negative controls were profiled using a TaqMan MicroRNA Array. A set of selected differentially expressed miRNAs were further analyzed in the patients at different perioperative periods, including preoperative, 1 week postoperative, and one month postoperative. The results showed that several serum miRNAs were differentially expressed in the cervical SCC patients compared with the negative controls, including miR-646, miR-141* and miR-542-3p. More importantly, we found that levels of specific serum miRNAs were deregulated in the pre- and postoperative stages, and these miRNAs could be useful for post-therapeutic monitoring of disease progression. Finally, we depicted a regulatory network of differentially expressed serum miRNAs, and many possible target genes were predicted in the estrogen-mediated signal pathways, supporting the hypothesis that cervical SCC is a hormone-associated gynecological disease.
Our study demonstrated that the circulating miRNAs miR-646, miR-141* and miR-542-3p could potentially serve as non-invasive biomarkers for cervical SCC. The levels of these specific miRNAs might be useful for the post-therapeutic monitoring of disease progression. This is the first report showing that circulating miRNAs could serve as biomarkers for the therapeutic intervention of cervical SCC.
PMCID: PMC3892020  PMID: 24405714
Circulating microRNA; Cervical SCC; Serum; Tumor biomarkers; Post-therapeutic monitoring
14.  Increased expression of miR-187 in human islets from individuals with type 2 diabetes is associated with reduced glucose-stimulated insulin secretion 
Diabetologia  2013;57:122-128.
Type 2 diabetes is characterised by progressive beta cell dysfunction, with changes in gene expression playing a crucial role in its development. MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression and therefore alterations in miRNA levels may be involved in the deterioration of beta cell function.
Global TaqMan arrays and individual TaqMan assays were used to measure islet miRNA expression in discovery (n = 20) and replication (n = 20) cohorts from individuals with and without type 2 diabetes. The role of specific dysregulated miRNAs in regulating insulin secretion, content and apoptosis was subsequently investigated in primary rat islets and INS-1 cells. Identification of miRNA targets was assessed using luciferase assays and by measuring mRNA levels.
In the discovery and replication cohorts miR-187 expression was found to be significantly increased in islets from individuals with type 2 diabetes compared with matched controls. An inverse correlation between miR-187 levels and glucose-stimulated insulin secretion (GSIS) was observed in islets from normoglycaemic donors. This correlation paralleled findings in primary rat islets and INS-1 cells where overexpression of miR-187 markedly decreased GSIS without affecting insulin content or apoptotic index. Finally, the gene encoding homeodomain-interacting protein kinase-3 (HIPK3), a known regulator of insulin secretion, was identified as a direct target of miR-187 and displayed reduced expression in islets from individuals with type 2 diabetes.
Our findings suggest a role for miR-187 in the blunting of insulin secretion, potentially involving regulation of HIPK3, which occurs during the pathogenesis of type 2 diabetes.
Electronic supplementary material
The online version of this article (doi:10.1007/s00125-013-3089-4) contains peer-reviewed but unedited supplementary material, which is available to authorised users.
PMCID: PMC3855472  PMID: 24149837
Glucose-stimulated insulin secretion; HIPK3; Islets; MicroRNA; Type 2 diabetes
15.  Expression profiling of cancerous and normal breast tissues identifies microRNAs that are differentially expressed in serum from patients with (metastatic) breast cancer and healthy volunteers 
MicroRNAs (miRNAs) are a group of small noncoding RNAs involved in the regulation of gene expression. As such, they regulate a large number of cellular pathways, and deregulation or altered expression of miRNAs is associated with tumorigenesis. In the current study, we evaluated the feasibility and clinical utility of circulating miRNAs as biomarkers for the detection and staging of breast cancer.
miRNAs were extracted from a set of 84 tissue samples from patients with breast cancer and eight normal tissue samples obtained after breast-reductive surgery. After reverse transcription and preamplification, 768 miRNAs were profiled by using the TaqMan low-density arrays. After data normalization, unsupervised hierarchical cluster analysis (UHCA) was used to investigate global differences in miRNA expression between cancerous and normal samples. With fold-change analysis, the most discriminating miRNAs between both tissue types were selected, and their expression was analyzed on serum samples from 20 healthy volunteers and 75 patients with breast cancer, including 16 patients with untreated metastatic breast cancer. miRNAs were extracted from 200 μl of serum, reverse transcribed, and analyzed in duplicate by using polymerase chain reaction (qRT-PCR).
UHCA showed major differences in miRNA expression between tissue samples from patients with breast cancer and tissue samples from breast-reductive surgery (P < 0.0001). Generally, miRNA expression in cancerous samples tends to be repressed when compared with miRNA expression in healthy controls (P = 0.0685). The four most discriminating miRNAs by fold-change (miR-215, miR-299-5p, miR-411, and miR-452) were selected for further analysis on serum samples. All miRNAs at least tended to be differentially expressed between serum samples from patients with cancer and serum samples from healthy controls (miR-215, P = 0.094; miR-299-5P, P = 0.019; miR-411, P = 0.002; and miR-452, P = 0.092). For all these miRNAs, except for miR-452, the greatest difference in expression was observed between serum samples from healthy volunteers and serum samples from untreated patients with metastatic breast cancer.
Our study provides a basis for the establishment of miRNAs as biomarkers for the detection and eventually staging of breast cancer through blood-borne testing. We identified and tested a set of putative biomarkers of breast cancer and demonstrated that altered levels of these miRNAs in serum from patients with breast cancer are particularly associated with the presence of metastatic disease.
PMCID: PMC3496152  PMID: 22353773
16.  Identification and Pathway Analysis of microRNAs with No Previous Involvement in Breast Cancer 
PLoS ONE  2012;7(3):e31904.
microRNA expression signatures can differentiate normal and breast cancer tissues and can define specific clinico-pathological phenotypes in breast tumors. In order to further evaluate the microRNA expression profile in breast cancer, we analyzed the expression of 667 microRNAs in 29 tumors and 21 adjacent normal tissues using TaqMan Low-density arrays. 130 miRNAs showed significant differential expression (adjusted P value = 0.05, Fold Change = 2) in breast tumors compared to the normal adjacent tissue. Importantly, the role of 43 of these microRNAs has not been previously reported in breast cancer, including several evolutionary conserved microRNA*, showing similar expression rates to that of their corresponding leading strand. The expression of 14 microRNAs was replicated in an independent set of 55 tumors. Bioinformatic analysis of mRNA targets of the altered miRNAs, identified oncogenes like ERBB2, YY1, several MAP kinases, and known tumor-suppressors like FOXA1 and SMAD4. Pathway analysis identified that some biological process which are important in breast carcinogenesis are affected by the altered microRNA expression, including signaling through MAP kinases and TP53 pathways, as well as biological processes like cell death and communication, focal adhesion and ERBB2-ERBB3 signaling. Our data identified the altered expression of several microRNAs whose aberrant expression might have an important impact on cancer-related cellular pathways and whose role in breast cancer has not been previously described.
PMCID: PMC3306365  PMID: 22438871
17.  Imprinted DLK1-DIO3 region of 14q32 defines a schizophrenia-associated miRNA signature in peripheral blood mononuclear cells 
Molecular Psychiatry  2011;17(8):827-840.
MicroRNAs (miRNAs) regulate gene expression at the post-transcriptional level and are important for coordinating nervous system development and neuronal function in the mature brain. We have recently identified schizophrenia-associated alteration of cortical miRNA biogenesis and expression in post-mortem brain tissue with implications for the dysregulation of schizophrenia candidate genes. Although these changes were observed in the central nervous system, it is plausible that schizophrenia-associated miRNA expression signatures may also be detected in non-neural tissue. To explore this possibility, we investigated the miRNA expression profile of peripheral blood mononuclear cells (PBMCs) from 112 patients with schizophrenia and 76 non-psychiatric controls. miRNA expression analysis of total RNA conducted using commercial miRNA arrays revealed that 33 miRNAs were significantly downregulated after correction for multiple testing with a false discovery rate (FDR) of 0%, which increased to 83 when we considered miRNA with an FDR<5%. Seven miRNAs altered in microarray analysis of schizophrenia were also confirmed to be downregulated by quantitative real-time reverse transcription-polymerase chain reaction. A large subgroup consisting of 17 downregulated miRNAs is transcribed from a single imprinted locus at the maternally expressed DLK1-DIO3 region on chromosome 14q32. This pattern of differentially expressed miRNA in PBMCs may be indicative of significant underlying genetic or epigenetic alteration associated with schizophrenia.
PMCID: PMC3404364  PMID: 21727898
biomarker; gene silencing; microRNA; PBMCs; schizophrenia; 14q32
18.  MicroRNA-146a expresses in interleukin-17 producing T cells in rheumatoid arthritis patients 
Interleukin (IL)-17 is an important factor in rheumatoid arthritis (RA) pathogenesis. MicroRNA (miRNA)s are a family of non coding RNAs and associated with human diseases including RA. The purpose of this study is to identify the miRNAs in the differentiation of IL-17 producing cells, and analyze their expression pattern in the peripheral blood mononuclear cells (PBMC) and synovium from RA patients.
IL-17 producing cells were expanded from CD4+T cell. MiRNA microarray was performed to identify the miRNAs in the differentiation of IL-17 producing cells. Quantitative polymerase chain reaction was performed to examine the expression patterns of the identified miRNAs in the PBMC and synovium from RA and osteoarthritis (OA) patients. Double staining combining in situ hybridization and immunohistochemistry of IL-17 was performed to analyze the expression pattern of identified miRNA in the synovium.
Six miRNAs, let-7a, miR-26, miR-146a/b, miR-150, and miR-155 were significantly up regulated in the IL-17 producing T cells. The expression of miR-146a and IL-17 was higher than in PBMC in the patients with low score of Larsen grade and short disease duration. MiR-146a intensely expressed in RA synovium in comparison to OA. MiR-146a expressed intensely in the synovium with hyperplasia and high expression of IL-17 from the patients with high disease activity. Double staining revealed that miR-146a expressed in IL-17 expressing cells.
These results indicated that miR-146a was associated with IL-17 expression in the PBMC and synovium in RA patients. There is the possibility that miR-146a participates in the IL-17 expression.
PMCID: PMC2950393  PMID: 20840794
19.  Identification of Novel Epstein-Barr Virus MicroRNA Genes from Nasopharyngeal Carcinomas▿ † 
Journal of Virology  2009;83(7):3333-3341.
MicroRNAs (miRNAs) represent a conserved class of small noncoding RNAs that are found in all higher eukaryotes as well as some DNA viruses. miRNAs are 20 to 25 nucleotides in length and have important regulatory functions in biological processes such as embryonic development, cell differentiation, hormone secretion, and metabolism. Furthermore, miRNAs have been implicated in the pathology of various diseases, including cancer. miRNA expression profiles not only classify different types of cancer but also may even help to characterize distinct tumor stages, therefore constituting a valuable tool for prognosis. Here we report the miRNA profile of Epstein-Barr virus (EBV)-positive nasopharyngeal carcinoma (NPC) tissue samples characterized by cloning and sequencing. We found that all EBV miRNAs from the BART region are expressed in NPC tissues, whereas EBV miRNAs from the BHRF1 region are not found. Moreover, we identified two novel EBV miRNA genes originating from the BART region that have not been found in other tissues or cell lines before. We also identified three new human miRNAs which might be specific for nasopharyngeal tissues. We further show that a number of different cellular miRNAs, including miR-15a and miR-16, are up- or downregulated in NPC tissues compared to control tissues. We found that the tumor suppressor BRCA-1 is a target of miR-15a as well as miR-16, suggesting a miRNA role in NPC pathogenesis.
PMCID: PMC2655542  PMID: 19144710
20.  Fecal microRNAs as novel biomarkers for colon cancer screening 
Colorectal cancer (CRC) is the second leading cause of cancer-related deaths, but currently available non-invasive screening programs have achieved only a modest decrease in mortality. MicroRNAs (miRNAs) play important role in a wide array of biological processes and are commonly dysregulated in neoplasia. We aimed to evaluate the feasibility of fecal miRNAs as biomarkers for colorectal neoplasia screening.
Material and methods
Total RNA was extracted from freshly collected stool samples from 8 healthy volunteers and 29 FOBT collected feces from subjects with normal colonoscopies, colon adenomas and CRCs. miRNA expression analysis were performed with TaqMan qRT-PCR for a subset of miRNAs. Illumina miRNA microarray profiling was performed to evaluate the differences in expression patterns between normal colonic mucosa tissues and stool samples from healthy subjects.
We efficiently extracted miRNAs from stool specimens using our developed protocol. Data from independent experiments showed high reproducibility for miRNA extraction and expression. miRNA expression patterns were similar in stool specimens among healthy volunteers and reproducible in stool samples that were collected serially in time from the same individuals. miRNA expression profiles from 29 patients demonstrated higher expression of miR-21 and -106a in patients with adenomas and CRCs, compared with individuals free of colorectal neoplasia.
Our data indicate that miRNAs can be extracted from stool easily and reproducibly. The stools of patients with colorectal neoplasms have unique and identifiable patterns of miRNA expression.
Fecal miRNAs may be an excellent candidate for the development of a non-invasive screening test for colorectal neoplasms.
PMCID: PMC2901410  PMID: 20551304
microRNA; stool biomarker; colon cancer; cancer screening
21.  A Global Analysis of Evolutionary Conservation among Known and Predicted Gammaherpesvirus MicroRNAs▿ †  
Journal of Virology  2009;84(2):716-728.
MicroRNAs (miRNAs) are small, noncoding RNAs which posttranscriptionally regulate gene expression. The current release of the miRNA registry lists 16 viruses which encode a total of 146 miRNA hairpins. Strikingly, 139 of these are encoded by members of the herpesvirus family, suggesting an important role for miRNAs in the herpesvirus life cycle. However, with the exception of 7 miRNA hairpins known to be shared by Epstein-Barr virus (EBV) and the closely related rhesus lymphocryptovirus (rLCV), the known herpesvirus miRNAs show little evidence of evolutionary conservation. We have performed a global analysis of miRNA conservation among gammaherpesviruses which is not limited to family members known to encode miRNAs but includes also those which have not been previously analyzed. For this purpose, we have performed a computational prediction of miRNA candidates of all fully sequenced gammaherpesvirus genomes, followed by sequence/structure alignments. Our results indicate that gammaherpesvirus miRNA conservation is limited to two pairs of viral genomes. One is the already-known case of EBV and rLCV. These viruses, however, share significantly more miRNAs than previously thought, as we identified and experimentally verified 10 novel conserved as well as 7 novel nonconserved rLCV pre-miRNA hairpins. The second case consists of rhesus rhadinovirus (RRV), which is predicted to share at least 9 pre-miRNAs with the closely related Japanese macaque herpesvirus (JMHV). Although several other gammaherpesviruses are predicted to encode large numbers of clustered miRNAs at conserved genomic loci, no further examples of evolutionarily conserved miRNA sequences were found.
PMCID: PMC2798347  PMID: 19889779
22.  Expression of Viral MicroRNAs in Epstein-Barr Virus-Associated Gastric Carcinoma▿  
Journal of Virology  2006;81(2):1033-1036.
Epstein-Barr virus (EBV) is associated with about 6 to 16% of gastric carcinoma cases worldwide. Expression of the EBV microRNAs (miRNAs) was observed in B cells and nasopharyngeal carcinoma cells infected with EBV. However, it is not clear if the EBV miRNAs are expressed in EBV-associated gastric carcinomas (EBVaGCs). We found that BART miRNAs but not BHRF1 miRNAs were expressed in EBV-infected gastric carcinoma cell lines and the tumor tissues from patients as well as the animal model. The expression of viral miRNAs in EBVaGCs suggests that these EBV miRNAs may play important roles in the tumorigenesis of EBVaGCs.
PMCID: PMC1797424  PMID: 17079300
23.  Identification and Analysis of Expression of Novel MicroRNAs of Murine Gammaherpesvirus 68▿ †  
Journal of Virology  2010;84(19):10266-10275.
Murine gammaherpesvirus 68 (MHV-68) is closely related to Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) and provides a small-animal model with which to study the pathogenesis of gammaherpesvirus (γHV) infections. To completely explore the potential of the MHV-68 system for the investigation of γHV microRNAs (miRNAs), it would be desirable to know the number and expression patterns of all miRNAs encoded by MHV-68. By deep sequencing of small RNAs, we systematically investigated the expression profiles of MHV-68 miRNAs in both lytically and persistently infected cells. In addition to the nine known MHV-68 miRNAs, we identified six novel MHV-68 miRNA genes and analyzed the expression levels of all MHV-68 miRNAs. Furthermore, we also characterized the cellular miRNA expression signatures in MHV-68-infected versus noninfected NIH 3T3 fibroblasts and in 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-treated versus nontreated S11 cells. We found that mmu-mir-15b and mmu-mir-16 are highly upregulated upon MHV-68 infection of NIH 3T3 cells, indicating a potential role for cellular miRNAs during MHV-68 infection. Our data will aid in the full exploration of the functions of γHV miRNAs.
PMCID: PMC2937759  PMID: 20668074
24.  HIV-1 encoded candidate micro-RNAs and their cellular targets 
Retrovirology  2004;1:43.
MicroRNAs (miRNAs) are small RNAs of 21–25 nucleotides that specifically regulate cellular gene expression at the post-transcriptional level. miRNAs are derived from the maturation by cellular RNases III of imperfect stem loop structures of ~ 70 nucleotides. Evidence for hundreds of miRNAs and their corresponding targets has been reported in the literature for plants, insects, invertebrate animals, and mammals. While not all of these miRNA/target pairs have been functionally verified, some clearly serve roles in regulating normal development and physiology. Recently, it has been queried whether the genome of human viruses like their cellular counterpart also encode miRNA. To date, there has been only one report pertaining to this question. The Epstein-Barr virus (EBV) has been shown to encode five miRNAs. Here, we extend the analysis of miRNA-encoding potential to the human immunodeficiency virus (HIV). Using computer-directed analyses, we found that HIV putatively encodes five candidate pre-miRNAs. We then matched deduced mature miRNA sequences from these 5 pre-miRNAs against a database of 3' untranslated sequences (UTR) from the human genome. These searches revealed a large number of cellular transcripts that could potentially be targeted by these viral miRNA (vmiRNA) sequences. We propose that HIV has evolved to use vmiRNAs as a means to regulate cellular milieu for its benefit.
PMCID: PMC544590  PMID: 15601472
25.  The microRNA Signature in Response to Insulin Reveals Its Implication in the Transcriptional Action of Insulin in Human Skeletal Muscle and the Role of a Sterol Regulatory Element–Binding Protein-1c/Myocyte Enhancer Factor 2C Pathway 
Diabetes  2009;58(11):2555-2564.
Factors governing microRNA expressions in response to changes of cellular environment are still largely unknown. Our aim was to determine whether insulin, the major hormone controlling whole-body energy homeostasis, is involved in the regulation of microRNA expressions in human skeletal muscle.
We carried out comparative microRNA (miRNA) expression profiles in human skeletal muscle biopsies before and after a 3-h euglycemic-hyperinsulinemic clamp, with TaqMan low-density arrays. Then, using DNA microarrays, we determined the response to insulin of the miRNA putative target genes in order to determine their role in the transcriptional action of insulin. We further characterized the mechanism of action of insulin on two representative miRNAs, miR-1 and miR-133a, in human muscle cells.
Insulin downregulated the expressions of 39 distinct miRNAs in human skeletal muscle. Their potential target mRNAs coded for proteins that were mainly involved in insulin signaling and ubiquitination-mediated proteolysis. Bioinformatic analysis suggested that combinations of different downregulated miRNAs worked in concert to regulate gene expressions in response to insulin. We further demonstrated that sterol regulatory element–binding protein (SREBP)-1c and myocyte enhancer factor 2C were involved in the effect of insulin on miR-1 and miR-133a expression. Interestingly, we found an impaired regulation of miRNAs by insulin in the skeletal muscle of type 2 diabetic patients, likely as consequences of altered SREBP-1c activation.
This work demonstrates a new role of insulin in the regulation of miRNAs in human skeletal muscle and suggests a possible implication of these new modulators in insulin resistance.
PMCID: PMC2768160  PMID: 19720801

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